The major discovery of Project 1 in the last cycle was that multiple abnormalities in the medullary serotonergic (5-HT) system are associated with SIDS. We now envision SIDS as a complex and heterogeneous process which involves multiple transmitters in addition to 5-HT, multiple stressors acting simultaneously, and multiple genetic and environmental factors, including chronic hypoxia and male gender, augmenting the brainstem defects. Our Specific Aims are: 1) To determine the neurochemical anatomy of Y-aminobutyric acid (GABA) in the medulla in SIDS cases. We will determine the number and density of GABA neurons, percent of 5-HT neurons that co-localize with GABA, GABAA receptor binding, and cellular localization of GABAA receptor subunits in SIDS cases compared with controls adjusted for age. 2) To determine the neurochemical anatomy of SP-expressing neurons and NK1 receptors in the medulla in SIDS cases. We will determine the number and density of SP neurons, the percent of 5-HT neurons that co-localize SP, SP binding, and cellular localization of NK1 in SIDS cases compared to controls adjusted for age. We will also determine if the SIDS cases with 5-HT1A and putative GABAA receptor binding abnormalities are the same as those with SP binding abnormalities. 3) To determine the role of estrogen and testosterone in the medullary pathology of SIDS. We will test the hypotheses that: androgen receptor binding is elevated in SIDS cases (male and female) in the medullary 5-HT system and/or its projection sites compared to controls adjusted for age; high serum testosterone levels correlate with high androgen binding in the same SIDS cases; and male SIDS cases with high serum testosterone levels have lower 5-HT1A receptor binding than SIDS infants with low serum testosterone levels and controls. 4) To determine the role of chronic intermittent hypoxia in the medullary pathology in SIDS and the role of compromised responses to hypoxia due to a defect in 5-HTmediated plasticity. We will test the hypothesis that SIDS cases show a reduced hypoxic response in the hypoglossal nucleus compared to hypoxic cases, as reflected in reduced hypoxic markers, e.g., erythropoietin. This hypothesis is based on the idea that hypoxic cases are equipped with a normal ability to respond to hypoxia, whereas the SIDS cases have an impaired ability. We will then test the hypothesis that markers of 5-HT-mediated plasticity to intermittent hypoxia (BDNF, TrkB receptors) are reduced in SIDS cases compared to hypoxic cases in the hypoglossal nucleus. This finding would suggest an inability of the SIDS infants to adapt to intermittent hypoxia due to 5-HT raphe-related abnormalities and impaired long-term facilitation. The proposed studies build upon an emerging picture of a complex brainstem phenotype in SIDS based upon a unique database accrued over 20 years in our laboratory.